鲫鱼MBSP的定点突变及其在大肠杆菌中的表达
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- 英文论文题名：Site-directed mutate and recombinant expression of myofibrilbound serine proteinase from crucian carp
- 论文作者：梅雪娇 ; 刘光明 ; 曹敏杰 ; 杜翠红
- 英文论文作者：Mei Xuejiao ; Liu Guangming ; Cao Minjie ; Du Cuihong ; College of Food and Bioscience Engineering ; Jimei University ; Key laboratory of Marine functional food in Xiamen ; Marine functional food engineering technology center of Fujian Province ; National Joint Engineering Research Center for deep processing of aquatic products
- 年：2016
- 作者机构：集美大学食品与生物工程学院厦门市海洋功能食品重点实验室福建省海洋功能食品工程技术研究中心水产品深加工技术国家地方联合工程研究中心;
- 论文关键词：肌原纤维结合型丝氨酸蛋白酶 ; 基因定点突变 ; 大肠杆菌表达系统 ; 重组表达
- 英文论文关键词：myofibril-bound serine proteinase ; site-directedmutate ; E.COLI expression system ; recombinant expression
- 会议召开时间：2016-11-09
- 会议录名称：中国食品科学技术学会第十三届年会论文摘要集
- 英文会议录名称：Abstracts of the 13th Annual Meeting of CIFST
- 语种：中文
- 分类号：Q55;Q78
- 学会代码：ZGSP
- 会议名称：中国食品科学技术学会第十三届年会
- 会议地点：中国北京
- 主办单位：中国食品科学技术学会
- 学会名称：中国食品科学技术学会
- 页数：2
- 文件大小：104k
- 原文格式：O
- 会议级别：全国
摘要
鲫鱼肌原纤维结合型丝氨酸蛋白酶(MBSP)是一种底物特异性类似胰蛋白酶的热稳定性蛋白酶,可用于水解蛋白以制备生物活性肽。如果能在保持其热稳定性的基础上,改变其底物特异性,将会获得一种新型的热稳定性蛋白酶。本实验采用重叠延伸PCR技术对MBSP基因进行定点突变,首先将成熟MBSP氨基酸序列中170位的Asp分别突变成Ser和Thr,获得两个突变体D170S和D170T基因;然后再将突变体D170T中203位的Gly突变成Ala,获得双突变体D170T/G203A基因。采用基因重组技术,借助大肠杆菌表达载体pV29wt将以上三个突变体基因分别转入大肠杆菌为表达宿主BL21(DE3)中,获得鲫鱼MBSP突变体的表达菌株。利用IPTG对重组菌株进行诱导表达,采用SDS-PAGE和Westernblot对重组蛋白进行鉴定。本实验为研究不同突变位点对MBSP底物特异性的影响奠定了基础。
Myofibril-bound serine proteinase(MBSP) from crucian carp is a kind of thermal stability protease which its substrate specificity similar as trypsin protease,it be used for protein hydrolysate in the preparation of bioactive peptides.If we can keep base on the thermal stability,changing the substrate specificity,it will be a new type of the thermal stability of protease.This experiment adopts the overlap extension PCR technology to site-directed MBSP gene mutations,making mature MBSP amino acid sequence of 170 site Asp mutate respectively into Ser and Thr firstly,get two mutant D170 S and D170 T genes;Then mutate D170 Ts 203 site Gly mutant into in Ala,get double mutant D170T/G203 A genes.Using gene recombination technology,with the aid of E.coli expression vector pV29 wt putting the above three mutant genes into E.coli host BL21(DE3),obtain the expression of crucian carp MBSP mutant strains.Using IPTG to induce expression of recombinant strains,using SDS-PAGE and western blot on recombinant protein identification.This experiment to study different mutations site affect MBSP substrate specificity laid a foundation.
Myofibril-bound serine proteinase(MBSP) from crucian carp is a kind of thermal stability protease which its substrate specificity similar as trypsin protease,it be used for protein hydrolysate in the preparation of bioactive peptides.If we can keep base on the thermal stability,changing the substrate specificity,it will be a new type of the thermal stability of protease.This experiment adopts the overlap extension PCR technology to site-directed MBSP gene mutations,making mature MBSP amino acid sequence of 170 site Asp mutate respectively into Ser and Thr firstly,get two mutant D170 S and D170 T genes;Then mutate D170 Ts 203 site Gly mutant into in Ala,get double mutant D170T/G203 A genes.Using gene recombination technology,with the aid of E.coli expression vector pV29 wt putting the above three mutant genes into E.coli host BL21(DE3),obtain the expression of crucian carp MBSP mutant strains.Using IPTG to induce expression of recombinant strains,using SDS-PAGE and western blot on recombinant protein identification.This experiment to study different mutations site affect MBSP substrate specificity laid a foundation.
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